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. 2000 Dec;59(12):975–981. doi: 10.1136/ard.59.12.975

Anti-ribosomal P protein antibodies detected by immunoblotting in patients with connective tissue diseases: their specificity for SLE and association with IgG anticardiolipin antibodies

A Ghirardello 1, A Doria 1, S Zampieri 1, R Gerli 1, E Rapizzi 1, P Gambari 1
PMCID: PMC1753043  PMID: 11087701

Abstract

OBJECTIVE—To assess the prevalence and clinical and serological associations of anti-ribosomal P protein antibodies (anti-P antibodies) in patients with connective tissue diseases (CTDs) and investigate the immunobiological nature of autoantibody clustering in which anti-P antibodies play a part.
METHODS—IgG anti-P antibodies in the sera of 267 patients with CTDs and 31 healthy subjects were analysed by immunoblotting performed on cytoplasmic extract of Raji cells. 60 patients with systemic lupus erythematosus (SLE), 32 systemic sclerosis, 46 primary Sjögren's syndrome, 16 poly/dermatomyositis, 11 rheumatoid arthritis, 8 undifferentiated CTD, 72 overlap CTD, and 22 primary antiphospholipid syndrome were studied. Anti-P antibodies were affinity purified by elution from nitrocellulose bound antigen and tested by ELISA for their binding activity to cardiolipin.
RESULTS—Anti-P antibodies were detected in 16 (6%) patients and in none of the controls: 12/60 SLE (20%) and 4/80 undifferentiated/overlap patients with CTD (5%). A close association of IgG antibodies with P proteins and with cardiolipin was seen in lupus sera (p=0.0009, odds ratio 18.33). Anti-P antibodies from 9 of 12 anti-P lupus serum samples could be affinity purified and none of the affinity purified fractions cross reacted with ELISA plate coated cardiolipin.
CONCLUSIONS—Anti-P immunoreactivity is a specific marker of SLE and lupus-like disease and its detection is recommended as a powerful diagnostic tool. Anti-P antibodies are strongly clustered with IgG anticardiolipin antibodies in lupus sera, even if they are independently elicited. This suggests that their cognate autoantigens play a part in a common pathogenetic pathway in SLE.



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Figure 1  .

Figure 1  

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Immunoblot analysis of serum samples from healthy donors on cytoplasmic extract of Raji cells resolved on 15% SDS-PAGE. Molecular weight markers are indicated in kilodaltons on the right.

Figure 2  .

Figure 2  

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Analysis of anti-P positive SLE sera (lanes 1-12) on a cytoplasmic blot. Immunoblot was interpreted as positive with concomitant lines corresponding to 38 kDa, 19 kDa, and 17 kDa bands. Control sera: (a) anti-P prototype serum; (b) anti-mitochondrial protein serum; (c) anti-La/SSB + anti-Ro/SSA 52 kDa serum; (d) anti-Jo-1 serum; (e) control serum from a healthy donor showing the background band typically present in Raji cell extracts. Molecular weight markers are indicated in kilodaltons on the right.

Figure 3  .

Figure 3  

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Anti-P antibody detected by immunoblotting: sensitivity for systemic lupus erythematosus (SLE) and undifferentiated/overlap connective tissue diseases (UCTD) diagnosis and its specificity in healthy subjects and CTDs other than SLE or UCTD.

Figure 4  .

Figure 4  

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Association of anti-P and antiphospholipid (IgG anticardiolipin antibody (aCL), IgM aCL, lupus anticoagulant (LA)) responses in patients with systemic lupus erythematosus.

Figure 5  .

Figure 5  

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Immunoblot with anti-P serum from a patient with systemic lupus erythematosus and affinity purified fractions (material concentrated or filtrated on Centricon 50 tubes). (A) Anti-P serum after immunoabsorption on a negative control protein region corresponding to 45 kDa; (B) the same anti-P serum after immunoabsorption on the regions corresponding to the P proteins of 38, 19, and 17 kDa; (C) affinity purified fraction on the negative control protein region (a, concentrated; b, filtrated); (D) affinity purified fraction on the P proteins (a, concentrated; b, filtrated). Molecular weight markers are indicated in kilodaltons on the right.

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